Protein-protein interactions are responsible for key biological functions such as cell division and enzyme regulation and are involved, for example, in the assembly of enzyme subunits; in antigen-antibody reactions; in forming the supramolecular structures of ribosomes, filaments, and viruses; in molecular transport; and in the interaction of receptors on cell surfaces with growth factors and hormones. It is also through protein-protein interactions that oncogene expression results in neoplastic transformation. For example, some oncogenes encode protein kinases whose enzymatic activity on cellular target proteins leads to the cancerous state.
In addition to using well known biochemical techniques to study protein-protein interactions, a method for detecting protein-protein interactions using a genetic system is described in U.S. Pat. No. 5,283,173. This two hybrid genetic system is capable of detecting proteins that interact with a known protein, determining which domains of the proteins interact, and providing the genes for newly identified interacting proteins. The two hybrid system of the '173 patent detects protein-protein interactions using transcriptional activation of a reporter gene as an assay mechanism.
Transcription can be activated through the use of two functional domains of a transcription activation moiety: a domain or sequence of amino acids that recognizes and binds to a specific site or sequence of nucleotides on a target DNA, i.e. a reporter gene; and a domain (sequence of amino acids) that is capable of activating transcription of the DNA when physically associated with the DNA-binding domain and is necessary for activation of the target gene. See Keegan, et al., Science, 231, 669-704 (1986); Ma and Ptashne, Cell, 48, 847-853 (1987). The two functional domains may be derived from a single transcription activation protein. Alternatively, it has been shown that these two functions can also reside on separate proteins (McKnight et al. Proc. Natl. Acad. Sci. USA 89, 7061-7065 (1987); Curran et al. 55, 395-397 (1988). The transcription activation domains may also be derived from synthetic DNA-binding and transcription activation proteins.
Transcriptional activation has been studied, for example, using the Ga14 protein of the yeast Saccharomyces cerevisiae. The Ga14 protein is a single transcriptional activation protein required for the expression of genes encoding enzymes of galactose utilization (Johnston, Microbiol. Rev., 51,458-476 (1987). Such a system is well known. See U.S. Pat. No. 5,283,173.
A disadvantage of studying protein-protein interactions using certain host cells, such as yeast, for the two hybrid system is that interactions mediated by post-translational modifications, such as tyrosine phosphorylation, cannot be detected. Especially limiting has been the inability of such a two hybrid system to detect interactions which are dependent on specific post-translational modifications which are not employed by the host cell into which the hybrid genes have been introduced.
Accordingly, it is an object of the present invention to provide an improved genetic method for the detection of protein-protein interactions, especially those protein interactions requiring specific post-translational modifications, such as the phosphorylation of tyrosine residues which is a critical step in the signal transduction pathways of activated cell-surface receptors.
Another object of the invention is to provide the regulated expression of proteins involved in protein-protein interactions as it relates to this system.
It is a further object of the present invention to provide a method for identifying amino acid residues which are critical for protein-protein interactions to occur.
Another object of the present invention is to provide a method for the identification of novel proteins which can be post-translationally modified.
It is a further object of the present invention to provide a method for the identification of novel proteins which bind to post-translationally modified proteins.
Another object of the present invention is to provide a method of identifying novel proteins that can post-translationally modify other proteins.
It is a further object of the invention to provide a method for the identification of molecules which inhibit protein-protein interactions, as directed by proteins capable of post-translationally modifying other proteins, proteins capable of being post-translationally modified by other proteins or proteins capable of binding to post-translationally modified proteins.